A *Lactobacillus plantarum* YYS-EN1 producing cellulase, pectinase, tanninase, and phytase and its applications

Abstract

This invention belongs to the field of microbial technology, specifically relating to a *Lactobacillus plantarum* YYS-EN1 that produces cellulase, pectinase, tanninase, and phytase, and its applications. This *Lactobacillus plantarum* YYS-EN1 is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 28532. The *Lactobacillus plantarum* YYS-EN1 provided by this invention possesses the ability to produce cellulase, pectinase, tanninase, and phytase, and can degrade plant cellulose and pectin, and decompose tannins and phytic acid. When applied to fermented products, it can reduce bitterness and improve product clarity. It can be widely used in the production of fermented products, such as fermented plant-derived nutritional powders and fermented plant-derived polypeptide powders, and can also be used in probiotic products and functional foods. It colonizes the human intestinal tract, improving gut microbiota structure and promoting digestion and absorption.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a plant lactobacillus YYS-EN1 that produces cellulase, pectinase, tanninase and phytase, and its applications. Background Technology

[0002] Grains, bran, fruits, and vegetables are rich in dietary fiber such as cellulose, pectin, and tannins, which are generally difficult to hydrolyze. Their rigid crystalline structure makes production challenging; during product processing, this not only increases processing steps and reduces yield but can also diminish the product's nutritional value to some extent. Physical and chemical degradation methods involve extreme conditions, long cycles, and are prone to secondary pollution. Therefore, microbial degradation has become the most effective, healthy, and environmentally friendly method.

[0003] Cellulase and pectinase hydrolyze plant crude fiber, facilitating the dissolution and release of substances such as starch and protein, thus aiding nutrient absorption and increasing product value. They are widely used in the extraction and clarification of fruits and vegetables, and in the fermentation of tea and coffee. Tanninase and phytase degrade tannins and phytic acid, improving the pressing and color of fruit juices and vegetables, and enhancing the flavor and color of wine.

[0004] In conclusion, cellulase, pectinase, tanninase, and phytase have broader application prospects in fermented plant-based beverages, fermented plant-derived nutritional powders, fermented plant-derived polypeptide powders, and fermented plant-derived protein powders.

[0005] Chinese invention patent application number CN202110864302.3, published on 20230203, discloses a method for preparing cellulase fermentation broth using a Streptomyces strain GZUIRF-Y1, and its application. The cellulase activity in the fermentation broth of this strain reaches 111.0 U·mL. -1 In this technology, the strains that degrade cellulose are molds.

[0006] Chinese invention patent application number CN202111637611.3, published on April 12, 2022, discloses a strain of Aspergillus oryzae and its application in high-salt, high-nitrogen fermented foods. The strain has a cellulase activity of 18.6 U·g. -1 The acid pectinase activity was 33.1 U·g. -1 In this technology, the strains that degrade cellulose are molds.

[0007] The aforementioned molds are capable of producing cellulase and / or pectinase, but do not simultaneously produce cellulase, pectinase, tanninase, or phytase. Furthermore, currently, most strains that degrade cellulose, pectin, tannins, and phytic acid are molds or Bacillus species, which contain few beneficial bacteria and therefore cannot be used in food fermentation.

[0008] In summary, how to develop natural probiotics that produce cellulase, pectinase, tanninase, and phytase is precisely the technical problem that this field is committed to solving. Summary of the Invention

[0009] To address the shortcomings of the prior art mentioned in the background section, this invention provides a *Lactobacillus plantarum* YYS-EN1 strain. This *Lactobacillus plantarum* YYS-EN1 is a natural strain capable of producing cellulase, pectinase, tanninase, and phytase. It can degrade plant cellulose and pectin, decompose tannins and phytic acid, reduce bitterness, and improve product clarity. It is widely used in the production of fermented products, such as fermented plant-derived nutritional powders and fermented plant-derived polypeptide powders. The technical solution is as follows:

[0010] The *Lactobacillus plantarum* YYS-EN1 provided by this invention has the following Latin scientific name: Lactiplantibacillus plantarum It was deposited on September 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28532.

[0011] This strain, *Lactobacillus plantarum* YYS-EN1, was isolated from rice wine. Sequencing analysis revealed high homology between this strain and *Lactobacillus plantarum* through BLAST sequence comparison, leading to its naming as *Lactobacillus plantarum* YYS-EN1. *Lactobacillus plantarum* YYS-EN1 possesses the ability to produce cellulase, pectinase, tanninase, and phytase, demonstrating its capacity to degrade cellulose, pectin, tannins, and phytic acid.

[0012] This invention also provides the application of *Lactobacillus plantarum* YYS-EN1 as described above in the preparation of fermented products. The application of *Lactobacillus plantarum* YYS-EN1 in the preparation of fermented products can significantly reduce the content of cellulose and pectin in the products.

[0013] In some embodiments, the fermented products include, but are not limited to, fermented nutrient powder, fermented polypeptide powder, fermented protein powder, enzymes, and silage.

[0014] In some embodiments, the fermented nutrient powder includes fermented plant-derived nutrient powder; the fermented polypeptide powder includes fermented plant-derived polypeptide powder; and the fermented protein powder includes fermented plant-derived protein powder.

[0015] In some embodiments, the fermented plant-derived nutrient powder includes fermented corn nutrient powder.

[0016] The Lactobacillus plantarum YYS-EN1 is used in fermented corn nutrient powder products. It can ferment corn husks and corn germ, decompose cellulose and pectin, and reduce tannins and phytic acid, thereby enhancing the flavor and taste of corn nutrient powder.

[0017] The present invention also provides a method for producing cellulase, pectinase, tanninase and phytase: Lactobacillus paracasei YYS-EN2 as described above is inoculated into an enzyme-producing liquid culture medium and cultured at (30-40)℃ for 48-72h; the enzyme-producing liquid culture medium after fermentation is separated into solid and liquid components to obtain a supernatant; the supernatant is an enzyme solution containing cellulase and / or pectinase and / or tanninase and / or phytase.

[0018] The present invention also provides a freeze-dried product, the components of which include Lactobacillus plantarum YYS-EN1 as described above.

[0019] In some embodiments, the viable count of *Lactobacillus plantarum* YYS-EN1 in the freeze-dried product is (2–8) × 10⁻⁶. 11 CFU·g -1 .

[0020] This invention also provides a method for preparing a freeze-dried product, comprising the following preparation steps:

[0021] 1) Preparation of seed culture of Lactobacillus plantarum YYS-EN1;

[0022] 2) Seed culture expansion;

[0023] 3) Seed culture fermentation to obtain fermentation broth;

[0024] 4) Centrifuge the fermentation broth to obtain bacterial sludge;

[0025] 5) After mixing the bacterial sludge with the freeze-drying protectant, emulsify and embed it to obtain an emulsion;

[0026] 6) The emulsion is freeze-dried and pulverized to obtain freeze-dried powder of Lactobacillus plantarum YYS-EN1.

[0027] The present invention also provides a microbial agent, wherein the components of the microbial agent comprise the freeze-dried powder as described above, and / or, the components of the microbial agent comprise *Lactobacillus plantarum* YYS-EN1 as described above.

[0028] In some embodiments, the microbial agent further includes other ingredients, which are existing ingredients suitable for microbial agents, such as at least one of prebiotics, fillers, acidulants, solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, stabilizers, flow aids, flavoring agents, preservatives, coating materials, fragrances, anti-adhesion agents, binding agents, thickeners, and inclusion agents.

[0029] The present invention also provides the application of Lactobacillus plantarum YYS-EN1 as described above in the preparation of probiotic products.

[0030] This invention also provides the application of *Lactobacillus plantarum* YYS-EN1 as described above in the preparation of functional products, said functional products comprising at least one of the following functions:

[0031] (1) It has the ability to break down cellulose;

[0032] (2) It has the ability to decompose pectin;

[0033] (3) It has the ability to decompose tannins;

[0034] (4) It has the ability to decompose phytic acid.

[0035] In some embodiments, the functional product includes food and health products; Lactobacillus plantarum YYS-EN1 can be used to prepare food or health products.

[0036] Based on the above characteristics, the *Lactobacillus plantarum* YYS-EN1 provided by this invention has the following beneficial effects:

[0037] The *Lactobacillus plantarum* YYS-EN1 provided by this invention has the ability to produce cellulase, pectinase, tanninase and phytase, and can degrade plant cellulose, pectin, tannin and phytic acid.

[0038] It has the ability to produce cellulase, which can degrade cellulose and can be applied to the development of fermented plant-derived nutrient powders, silage products, etc.

[0039] It has the ability to degrade pectinase and can be widely used in the production of fruit juice, vegetables, wine, enzymes and other products. It can significantly reduce the pectin content in raw materials and improve the clarity and yield of products.

[0040] It has the ability to produce tannins and can be widely used in the production of wine, enzymes and other products. It can reduce the tannin content in wine and improve the taste of the product.

[0041] It has the ability to produce phytase, which can be widely used in flour and soybean food processing to improve the degradation rate of phytic acid and enhance its nutritional and commercial value.

[0042] In summary, the *Lactobacillus plantarum* YYS-EN1 provided by this invention produces cellulase, pectinase, tanninase, and phytase, and can be applied to various probiotic fermentation products, laying the foundation for providing green, healthy, high-nutritional-value, and highly bioavailable plant-based agricultural products. It can also be used in probiotic products and functional foods, colonizing the human gut and improving gut microbiota structure and promoting digestion and absorption. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 Colony diagram of Lactobacillus plantarum YYS-EN1 on MRS plate medium;

[0045] Figure 2 A graph showing the glucose standard curve;

[0046] Figure 3 The standard curve for D-galacturonic acid is shown in the figure.

[0047] Figure 4 The standard curve for gallic acid is shown in the figure.

[0048] Figure 5 This is a graph showing the standard curve for inorganic phosphorus. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] The solution of the present invention:

[0051] This invention provides a plant lactobacillus ( Lactiplantibacillus plantarum YYS-EN1:

[0052] Lactobacillus plantarum ( Lactiplantibacillus plantarum YYS-EN1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 25, 2023, with accession number CGMCC No. 28532.

[0053] Source: The *Lactobacillus plantarum* strain was isolated from rice wine. After sequencing analysis, the strain was found to be highly homologous to *Lactobacillus plantarum* by Blast sequence comparison, and was named *Lactobacillus plantarum* YYS-EN1.

[0054] Colony morphology: In MRS solid medium, colonies are milky white, round, and have smooth and neat edges.

[0055] Functions: It has the ability to produce cellulase and pectinase, which can degrade cellulose and pectin. It also has the ability to produce tanninase and phytase, which can break down tannins and phytic acid.

[0056] The present invention also provides an operational example of a method for producing cellulase and / or pectinase and / or tanninase and / or phytase:

[0057] Lactobacillus paracasei YYS-EN2 as described above was inoculated into an enzyme-producing liquid culture medium and cultured at (30-40) °C for 48-72 h. The enzyme-producing liquid culture medium after fermentation was separated into solid and liquid components to obtain a supernatant. The supernatant was an enzyme solution containing cellulase and / or pectinase and / or tanninase and / or phytase.

[0058] This invention also provides a method for preparing a freeze-dried product, comprising the following preparation steps:

[0059] 1) Preparation of seed culture of Lactobacillus plantarum YYS-EN1;

[0060] 2) Seed culture expansion;

[0061] 3) Seed culture fermentation to obtain fermentation broth;

[0062] 4) Centrifuge the fermentation broth to obtain bacterial sludge;

[0063] 5) After mixing the bacterial sludge with the freeze-drying protectant, emulsify and embed it to obtain an emulsion;

[0064] 6) The emulsion is freeze-dried and pulverized to obtain freeze-dried powder of Lactobacillus plantarum YYS-EN1.

[0065] This invention also provides an operational example of its application method in the production of corn nutrient powder:

[0066] Corn germ and corn husk are crushed, then treated with lipase and amylase, sterilized at high temperature, and then *Lactobacillus plantarum* YYS-EN1 bacterial powder and / or freeze-dried powder are added. After mixing and fermentation, the mixture is sterilized at high temperature and then dried and stored to obtain corn nutrient powder.

[0067] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1: Isolation and identification of Lactobacillus plantarum YYS-EN1

[0069] Filtering and separation:

[0070] Aseptic sampling was performed on rice wine using the plate spread method. 5g of rice wine sample was placed in a sterile homogenizing bag, and 45mL of 0.9% physiological saline was added. The mixture was then homogenized to obtain the sample. 100μL of the sample was then serially diluted 10-fold. -2 10 -3 10 -4 100 μL of the sample was spread onto MRS solid plates containing 2.5% CaCO3 and incubated upside down at 37°C for 24 h. Colonies with good growth and large calcium dissolution zones were selected and repeatedly isolated and purified by streak plating until single colonies were obtained. The isolated bacteria were screened to obtain strains that produced high levels of cellulase, pectinase, tanninase, and phytase. This isolated strain was named YYS-EN1 and stored in a bacterial culture library at -80°C with added glycerol.

[0071] The colony morphology of the isolated and purified Lactobacillus plantarum YYS-EN1 is as follows: in MRS solid medium, the colonies are milky white, round, and have smooth and neat edges.

[0072] The process for screening bacteria that produce high levels of cellulase, pectinase, tanninase, and phytase is as follows:

[0073] Screening of cellulase-producing strains:

[0074] 1. Screening medium (g / L): Sodium carboxymethyl cellulose (CMC-Na) 20g, peptone 10g, yeast extract 5g, sodium chloride 5g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.3g; Congo red 0.4g;

[0075] 2. Strain screening: The bacteria were inoculated into MRS liquid medium from a -80℃ freezer and cultured at 37℃ for 24h; then, 100 μL of culture medium was placed on a screening medium plate using the Oxford cup method and cultured at 37℃ for 48h. The diameter of the clear zone was observed, and strains with high cellulase production were selected based on the diameter of the clear zone. Cellulase activity was measured to screen out strains with high cellulase production.

[0076] Similarly, the screening of bacteria that produce high levels of pectinase, tanninase, and phytase was carried out sequentially, and the specific process was as follows:

[0077] Screening of pectinase-producing strains:

[0078] 1. Screening medium (gL-1): pectin 10g, Congo red 0.2g, peptone 1.5g, dipotassium hydrogen phosphate 4g, potassium dihydrogen phosphate 2g, ammonium sulfate 4g, magnesium sulfate heptahydrate 0.5g.

[0079] 2. Strain screening: The bacteria were inoculated into MRS liquid medium from a -80℃ freezer and cultured at 37℃ for 24h; then, 100μL of culture medium was placed on a screening medium plate using the Oxford cup method and cultured at 37℃ for 48h. The diameter of the clear zone was observed, and strains with high pectinase production were selected based on the diameter of the clear zone. Pectinase activity was measured to screen out strains with high pectinase production.

[0080] Screening of tannin-producing strains

[0081] 1. Screening medium (gL-1): Sodium nitrate 3 g, dipotassium hydrogen phosphate 1 g, potassium chloride 0.5 g, magnesium sulfate 0.5 g; bromophenol blue 0.04 g, tannic acid 10 g, sterilized separately.

[0082] 2. Strain Screening: Bacteria were inoculated into MRS liquid medium from a -80℃ freezer and incubated at 37℃ for 24 h. Then, using the Oxford cup method, 100 μL of the culture was placed on screening medium plates and incubated at 37℃ for 48 h. The screening plates contained tannic acid and bromophenol blue indicator. If the strain produced tanninase, it would hydrolyze the tannic acid in the medium to produce gallic acid, causing the bromophenol blue indicator around the colony to change from blue-purple to yellow, thus forming a distinct color-changing zone around the colony. High-tanninase-producing strains were selected based on the intensity and diameter of the color-changing zone, and tanninase activity was measured to screen for high-tanninase-producing strains.

[0083] Screening of phytase-producing strains

[0084] 1. Screening medium (g / L) -1 Ingredients: 15g glucose, 5.0g ammonium nitrate, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.3g ferrous sulfate, 0.3g manganese sulfate, 2g calcium phytate.

[0085] 2. Strain Screening: The bacteria were inoculated into MRS liquid medium from a -80℃ freezer and cultured at 37℃ for 24 hours. Then, using the Oxford cup method, 100 μL of the culture solution was placed on a screening medium plate and cultured at 37℃ for 48 hours. Phytase-producing strains were able to hydrolyze calcium phytate, forming a clear zone around the colony. High-yielding phytase-producing strains were selected based on the diameter of the clear zone, and phytase activity was measured to further screen for high-yielding phytase-producing strains.

[0086] The formula for MRS liquid medium is as follows: 10.0 g beef extract, 20.0 g glucose, 10.0 g tryptone, 5.0 g yeast extract, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 2.0 g ammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate, 0.25 g manganese sulfate monohydrate, 1.0 L deionized water, pH 6.5 (adding 2% agar makes it MRS solid medium).

[0087] Strain identification:

[0088] (1) Observation of fungal morphology

[0089] The screened and purified strain YYS-EN1 was subjected to Gram staining and catalase tests, and its physiological and biochemical indicators were measured. The test results were compared with those in Bergey's Manual of Systematic Bacteriology, 8th Edition, for preliminary identification of the bacterial species. Figure 1 As shown, the test results showed that the selected strain YYS-EN1 stained purple with Gram stain, indicating a positive result. It is rod-shaped, catalase-negative, and does not form spores.

[0090] (2) Molecular biological identification

[0091] DNA was extracted from YYS-EN1 according to the instructions of the bacterial DNA extraction kit and then amplified by PCR:

[0092] PCR amplification process: The 16S rDNA gene sequence was amplified using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'); PCR reaction system: 1 μL of 27F (10 μM), 1 μL of 1492R (10 μM), and 10X... EasyTag @ R Buffer(2.5 mM) 5μL, dNTPs (2.5 mM) 4μL, DNA template 1μL, EasyTag @ R DNA Polymerase (5 U / L) 0.3 μL, ddH2O 37.7 μL. PCR amplification program: 94℃ 5 min, 94℃ 30 s, 55℃ 30 s, 72℃ 90 s, 72℃ 10 min, cycle from step 2 to step 4 32 times, store at 4℃.

[0093] ③PCR product detection and sequencing analysis: The amplified product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The strain YYS-EN1 was identified as Bacillus plantarum.

[0094] The 16S rDNA gene sequence is as follows:

[0095]

[0096] Example 2: Preparation of Lyophilized Powder of Lactobacillus plantarum YYS-EN1

[0097] The activated *Lactobacillus plantarum* YYS-EN1 was inoculated at a rate of 3% (v / v) into a culture medium sterilized at 121°C for 15 min, and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C, 6000 rpm for 10 min, and the supernatant was discarded to obtain bacterial sludge. This sludge was then emulsified with a cryoprotectant for 15 min to obtain a bacterial solution with a concentration of 5 × 10⁻⁶. 10 CFU·mL -1 The emulsion was pre-frozen at -40°C for 4 hours and then freeze-dried at -35°C for 35 hours to obtain a viable bacterial count of 510 billion CFU·g. -1 Freeze-dried active Lactobacillus plantarum YYS-EN1 bacterial powder.

[0098] The culture medium used consisted of the following components (by weight percentage): 2.5% glucose, 2.5% soybean peptone, 1% yeast extract, 1% beef extract, 0.6% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.03% manganese sulfate, 0.1% Tween 80, and the balance being water. The protective agent was prepared at 50 g / L. -1 Skim milk powder, 10 g·L -1 Sucrose, 10 g·L -1 Trehalose and 10 g·L -1 The composition of L-glutamate sodium.

[0099] It should be noted that the protective agent can be an existing protective agent component or formulation, including but not limited to the above-described embodiments.

[0100] Example 3: Cellulase activity of Lactobacillus plantarum YYS-EN1

[0101] 1. Enzyme-producing medium (g / L) -1 Ingredients: Sodium carboxymethyl cellulose (CMC-Na) 20g, peptone 3g, yeast extract 0.5g, ammonium sulfate 2g, potassium dihydrogen phosphate 4g, magnesium sulfate 0.3g, calcium chloride 0.3g;

[0102] 2. Cellulase Activity Assay

[0103] (1) Definition of enzyme activity: Under the conditions of 50℃ and pH 4.5, the enzyme can decompose 10 g·L per minute. -1 The amount of enzyme required to produce 1 mg of glucose from sodium carboxymethyl cellulose (CMC-Na) substrate is defined as one unit of enzyme activity (U·mL). -1 ).

[0104] (2) Reagent preparation:

[0105] 0.1 mol·L-1 Acetic acid-sodium acetate buffer (pH 4.6): Add 0.1 mol·L⁻¹ -1 Sodium acetate solution and 0.1 mol·L -1 The acetic acid solution was mixed and the pH was adjusted to 4.5. The concentration was 0.1 mol·L⁻¹. -1 Preparation of acetic acid solution: Accurately weigh 6g of acetic acid, add distilled water, and dilute to a volumetric flask of 1000mL; 0.1mol·L -1 Preparation of sodium acetate solution: Accurately weigh 8.20 g of anhydrous sodium acetate, add distilled water, and dilute to a volumetric flask of 1000 mL;

[0106] Sodium carboxymethyl cellulose (CMC-Na) solution (10 g·L⁻¹) -1 ): Accurately weigh 1g of sodium carboxymethyl cellulose (CMC-Na) and add it to 100mL of acetate-sodium acetate buffer.

[0107] (3) Enzyme activity assay: Cellulase activity was determined by the DNS method.

[0108] Preheat a 1% sodium carboxymethyl cellulose (CMC-Na) solution in a 40°C water bath. Add 1 mL of enzyme solution to 1 mL of the sodium carboxymethyl cellulose solution and react at 40°C for 30 min (enzyme time). Then add 1 mL of 0.4 mol·L⁻¹ enzyme solution. -1 The reaction was terminated with NaOH, while the blank group was terminated with NaOH solution before the enzyme solution was added.

[0109] After the reaction was complete, 1 mL of the reaction solution was mixed with 1 mL of DNS (5-(dimethylamino)-1-naphthalenesulfonic acid hydrate) solution, and the mixture was reacted in a boiling water bath for 5 min to develop color. The reaction solution after color development was diluted 5 times, and the sample absorbance was measured at 540 nm using a spectrophotometer with distilled water as the reference. The enzyme activity was then calculated according to the formula.

[0110] In this embodiment, the crude enzyme solution is obtained as follows: *Lactobacillus plantarum* YYS-EN1 is inoculated into the enzyme-producing medium and cultured at 37°C for 48 hours. The culture medium is then centrifuged at 4°C, and the supernatant is collected as the crude enzyme solution. The crude enzyme solution is then diluted with the test enzyme solution at a certain ratio for enzyme activity determination. The inoculation amount of *Lactobacillus plantarum* YYS-EN1 is 2% (v / v) of the culture medium.

[0111] Result calculation method:

[0112] The formula for enzyme activity determination is: Enzyme activity = OD 540 ×n×1 / k×1 / 30;

[0113] In the formula, n is the dilution factor for enzyme solution testing (the dilution factor from crude enzyme solution to test enzyme solution), k is the slope of the fitted standard curve, and 30 is the enzyme-catalyzed reaction time.

[0114] The standard curve was plotted using 1 g·L⁻¹. -1 Preparation of standard solutions of different concentration gradients (0 g·L⁻¹) from glucose solution -1 0.2 g·L -1 0.4 g·L -1 0.6 g·L -1 0.8 g·L -1 1.0 g·L -1 Following the enzyme activity assay method, absorbance was measured at 540 nm. A linear standard curve was plotted with glucose concentration on the x-axis and OD value on the y-axis (see [reference needed]). Figure 2 )

[0115] The results showed that the cellulase activity of *Lactobacillus plantarum* YYS-EN1 was 55.21 ± 0.06 U·mL. -1 .

[0116] Example 4: Pectinase activity of Lactobacillus plantarum YYS-EN1

[0117] 1. Enzyme-producing medium (g / L) -1 ): Pectin 10g, glucose 10g, peptone 4g, dipotassium hydrogen phosphate 4g, potassium dihydrogen phosphate 2g, magnesium sulfate heptahydrate 0.5g;

[0118] 2. Pectinase activity assay

[0119] (1) Definition of enzyme activity: Under the conditions of 50℃ and pH 5.0, the enzyme can decompose 10 g·L per minute. -1 The amount of enzyme required to produce 1 mg of galacturonic acid from a pectin substrate is defined as one unit of enzyme activity (U·mL). -1 );

[0120] (2) Solution preparation:

[0121] 0.2 mol·L -1 Acetic acid-sodium acetate buffer (pH 4.8): Add 0.2 mol·L⁻¹ -1 Sodium acetate solution and 0.2 mol·L -1 Mix the acetic acid solution and adjust the pH to 5.0.

[0122] Among them, 0.2 mol·L -1 Acetic acid solution: Accurately weigh 12g of acetic acid, add distilled water, and dilute to 1000mL in a volumetric flask; 0.2mol·L⁻¹ -1 Sodium acetate solution: Accurately weigh 16.41 g of sodium acetate, add distilled water, and dilute to a volumetric flask of 1000 mL. Mix the solution and adjust the pH to 5.0.

[0123] Pectin substrate (10 g·L) -1 ): 1g of pectin is added to 100mL of acetate-sodium acetate buffer;

[0124] (3) Enzyme activity assay:

[0125] Preheat the pectin substrate in a 50°C water bath. Add 1 mL of enzyme solution to 1 mL of the preheated pectin substrate, shake well, and react in a 50°C water bath for 30 min. Terminate the reaction by adding 1 mL of 10% trichloroacetic acid solution. For the blank control group, add the stop solution before adding the enzyme solution. After the reaction, mix 1 mL of the reaction solution with 1 mL of DNS solution and react in a boiling water bath for 5 min to develop color. Dilute the color-developed reaction solution 5 times, zero the sample using distilled water as a reference, and measure the absorbance of the sample at 540 nm using a spectrophotometer. Calculate the enzyme activity according to the formula.

[0126] The crude enzyme solution was obtained as follows: Lactobacillus plantarum YYS-EN1 was inoculated into the enzyme-producing medium and cultured at 37°C for 48 hours. The culture solution was centrifuged at 4°C and the supernatant was collected, which is the crude enzyme solution. The crude enzyme solution was diluted with the test enzyme solution at a certain ratio for enzyme activity determination. The inoculation amount of Lactobacillus plantarum YYS-EN1 was 2% (v / v) of the culture medium.

[0127] Result calculation method:

[0128] The formula for enzyme activity determination is: Enzyme activity = OD 540 ×n×1 / k×1 / 30;

[0129] In the formula, n is the dilution factor for enzyme solution testing (the dilution factor from crude enzyme solution to test enzyme solution), k is the slope of the fitted standard curve, and 30 is the enzyme-catalyzed reaction time.

[0130] The standard curve was plotted using 1 g·L⁻¹. -1 Prepare standard solutions of different concentration gradients (0 g·L⁻¹) from D-galacturonic acid solution. -1 0.2 g·L -1 0.4 g·L -1 0.6 g·L -1 0.8 g·L -1 1.0 g·L -1 Following the enzyme activity assay method, absorbance was measured at 540 nm. A linear standard curve was plotted with D-galacturonic acid concentration on the x-axis and OD value on the y-axis (see [link to standard curve]). Figure 3 ).

[0131] The test results showed that the pectinase activity of *Lactobacillus plantarum* YYS-EN1 was 45.28 ± 0.07 U·mL. -1 .

[0132] Example 5: Tannin activity of Lactobacillus plantarum YYS-EN1

[0133] 1. Enzyme-producing medium (g / L) -1 Ingredients: 10g glucose, 3g sodium nitrate, 1g dipotassium hydrogen phosphate, 0.5g potassium chloride, 0.5g magnesium sulfate, 20g tannic acid.

[0134] 2. Tannin enzyme activity assay

[0135] (1) Definition of enzyme activity: Under the conditions of 30℃ and pH 5.0, the enzyme activity is 0.01 mol·L⁻¹ of substrate per minute. -1 The amount of enzyme required to produce 1 mg of gallic acid from propyl gallate is defined as one unit of enzyme activity (U·mL). -1 );

[0136] (2) Solution preparation:

[0137] 0.1 mol·L -1 Citrate-sodium citrate buffer (pH 5.0): Add 0.1 mol·L⁻¹ -1 Citric acid solution and 0.1 mol·L -1 The sodium citrate solution was mixed and the pH was adjusted to 5.0. The solution contained 0.1 mol·L⁻¹ -1 For the citric acid solution, accurately weigh 19.21 g of citric acid and dilute to a volumetric flask with distilled water to a final volume of 1000 mL; 0.1 mol·L⁻¹ -1 For the sodium citrate solution, accurately weigh 29.41 g of sodium citrate, dissolve it in distilled water, and dilute to a volumetric flask of 1000 mL.

[0138] propyl gallate solution (0.01 mol·L⁻¹) -1 Accurately weigh 0.21 g of propyl gallate and dilute to 100 mL in a volumetric flask with citric acid buffer solution.

[0139] Methanol-Rhodanine Solution: Accurately weigh 0.667 g of rhodanine and dilute to 100 mL in a volumetric flask with methanol.

[0140] Gallic acid standard solution (5 mg·L) -1 Accurately weigh 0.5g of gallic acid and dilute to 100mL in a volumetric flask with methanol.

[0141] (3) Enzyme activity assay:

[0142] Preheat the propyl gallate solution in a 30°C water bath. Add 0.5 mL of the enzyme solution to 0.5 mL of the preheated propyl gallate solution, shake well, and react in a 30°C water bath for 5 min. Add 0.6 mL of methanol-rhodanine, and react in a 30°C water bath for 5 min. Add 0.8 mL of KOH, and react in a 30°C water bath for 5 min. Add 7.6 mL of distilled water, and react in a 30°C water bath for 10 min. Zero the sample using distilled water as a reference, and measure the absorbance at 520 nm using a spectrophotometer. Calculate the enzyme activity according to the formula. For the blank control group, add the stop solution KOH before adding the enzyme solution.

[0143] The crude enzyme solution was obtained as follows: Lactobacillus plantarum YYS-EN1 was inoculated into the enzyme-producing medium and cultured at 37°C for 48 hours. The culture medium was centrifuged at 4°C and the supernatant was collected, which was the crude enzyme solution. The crude enzyme solution was diluted with the test enzyme solution at a certain ratio for enzyme activity determination. The inoculation amount of Lactobacillus plantarum YYS-EN1 was 2% (v / v) of the culture medium.

[0144] Result calculation method:

[0145] The formula for enzyme activity determination is: Enzyme activity = OD 520 ×n×1 / k×1 / 5×1 / 0.5;

[0146] In the formula, n is the dilution factor for enzyme solution testing (the dilution factor from crude enzyme solution to test enzyme solution), k is the slope of the fitted standard curve, 5 is the enzyme-catalyzed reaction time, and 1 / 0.5 is converted into 1 mL of enzyme solution.

[0147] The standard curve was plotted using 5 mg·L⁻¹. -1 Standard solutions of different concentration gradients (0 mg·L⁻¹) were prepared from gallic acid solution. -1 1 mg·L -1 2 mg·L -1 3 mg·L -1 4 mg·L -1 5 mg·L -1 Following the enzyme activity assay method, absorbance was measured at 520 nm. A linear standard curve was plotted with gallic acid concentration on the x-axis and OD value on the y-axis (see [reference]). Figure 4 );

[0148] The results showed that the tanninase activity of *Lactobacillus plantarum* YYS-EN1 was 27.55 ± 0.03 U·mL. -1 .

[0149] Example 6: Phytase activity of Lactobacillus plantarum YYS-EN1

[0150] 1. Enzyme-producing medium (g / L) -1): Peptone 3g, glucose 15g, magnesium sulfate 0.5g, manganese sulfate 0.03g, ferrous sulfate 0.03g.

[0151] 2. Phytase activity assay

[0152] (1) Definition of enzyme activity: 1 ml of liquid enzyme decomposes 50 mmol·L per minute at 37℃ and pH 5.5. -1 Sodium phytate substrate produces 1 μmol of inorganic phosphorus, which is equivalent to one unit of phytase activity, expressed in U·mL. -1 .

[0153] (2) Reagent preparation:

[0154] 0.2 mol·L -1 Acetic acid-sodium acetate buffer (pH 5.5): Add 0.2 mol·L⁻¹ -1 Acetic acid solution and 0.2 mol·L -1 Sodium acetate solution was mixed and the pH was adjusted to 5.5. The sodium acetate solution was 0.2 mol·L⁻¹. -1 Acetic acid solution: 12g of acetic acid was diluted with distilled water to a volumetric flask to a final volume of 1000 mL; 0.2mol·L⁻¹ -1 Sodium acetate solution: 16.406 g of sodium acetate was dissolved in distilled water and diluted to a volumetric flask of 1000 mL.

[0155] Sodium phytate solution (75 mmol·L) -1 Accurately weigh 0.69 g of sodium phytate, dissolve it in acetic acid-sodium acetate buffer, and dilute to a volumetric flask of 100 mL.

[0156] Nitric acid solution: Nitric acid: Water (v / v) = 1:2;

[0157] Ammonium molybdate solution (100 g / L): Accurately weigh 10 g of ammonium molybdate, add 1 mL of 25% (v / v) ammonia solution, and dilute to 100 mL with distilled water in a volumetric flask.

[0158] Ammonium metavanadate solution (2.35 g / L): Accurately weigh 0.235 g of ammonium metavanadate, dissolve it in distilled water, add 2 mL of nitric acid solution, and dilute to 100 mL with distilled water in a volumetric flask. Store in the dark and prepare fresh before use.

[0159] Colorimetric solution (stop solution): Nitric acid solution: Ammonium molybdate solution: Ammonium metavanadate solution (v / v) = 2:1:1. Store protected from light and prepare fresh before use.

[0160] (3) Enzyme activity assay:

[0161] First, mix 1.8 mL of acetate buffer with 0.2 mL of the reaction solution (i.e., enzyme solution), preheat in a water bath at 37°C for 5 min, then add 4 mL of sodium phytate and mix well. Incubate in a water bath at 37°C for 30 min. Finally, add 4 mL of colorimetric solution (stop solution) and mix well. Zero the sample using distilled water as a reference and measure the absorbance at 415 nm using a spectrophotometer. Calculate the enzyme activity according to the formula. For the blank control group, add the stop solution before adding the enzyme solution.

[0162] The crude enzyme solution was obtained as follows: Lactobacillus plantarum YYS-EN1 was inoculated into the enzyme-producing medium and cultured at 37°C for 48 hours. The culture solution was centrifuged at 4°C and the supernatant was collected, which was the crude enzyme solution. The crude enzyme solution was diluted with enzyme solution for testing at a certain ratio for enzyme activity determination. The inoculation amount of Lactobacillus plantarum YYS-EN1 was 2% (v / v) of the culture medium.

[0163] Result calculation method:

[0164] The formula for enzyme activity determination is: Enzyme activity = OD 415 ×n×1 / k×1 / 30×1 / 0.2×10 3 ;

[0165] In the formula, n is the dilution factor for enzyme solution testing (the dilution factor from crude enzyme solution to test enzyme solution), k is the slope of the fitted standard curve, 30 is the enzyme-catalyzed reaction time, 1 / 0.2 is converted to 1 mL of enzyme solution, and 10 3 This is a conversion factor for the concentration of inorganic phosphate ions.

[0166] The standard curve was prepared by using potassium dihydrogen phosphate stock solution to prepare inorganic phosphorus standard solutions of different concentrations (0 mmol·L⁻¹). -1 10 mmol·L -1 20 mmol·L -1 30 mmol·L -1 40 mmol·L -1 50 mmol·L -1 OD was measured according to the enzyme activity assay method. 415 A linear standard curve was plotted with absorbance values, inorganic phosphate ion concentration on the x-axis and OD values ​​on the y-axis (see [reference]). Figure 5 ).

[0167] The results showed that the cellulase activity of Lactobacillus plantarum YYS-EN1 was 19.79±0.04 U / mL.

[0168] Example 7: Application of Lactobacillus plantarum YYS-EN1 in the preparation of fermented corn nutrient powder

[0169] Corn germ and corn bran were crushed, then treated with lipase and amylase, sterilized at high temperature, and then 3% (by weight) of *Lactobacillus plantarum* YYS-EN1 bacterial powder and / or freeze-dried powder were added. After mixing thoroughly, the mixture was fermented at 37℃ for 72 hours. After fermentation, it was sterilized at high temperature and dried at 60℃ to produce corn nutritional powder. *Lactobacillus plantarum* YYS-EN1 fermentation of corn bran and corn germ decomposes cellulose and pectin and reduces tannins and phytic acid, enhancing the flavor and texture of the corn nutritional powder. Specific flavor results before and after fermentation are detailed in Table 1 below:

[0170] Table 1

[0171]

[0172] Example 8: Application of Lactobacillus plantarum YYS-EN1 in the preparation of fermented traditional Chinese medicine

[0173] Polygonatum sibiricum, ginseng (artificially cultivated), wolfberry, mulberry, and red dates were pulverized and added to the mixture at weight percentages of 0.5%, 0.3%, 1%, 1.5%, and 2%, respectively. The mixture was thoroughly mixed, boiled, and kept at a gentle simmer for 1 hour. Then, 0.03% of *Lactobacillus plantarum* YYS-EN1 bacterial powder and / or freeze-dried powder and 0.02% of *Lactobacillus plantarum* BXM2 were added, mixed thoroughly, and fermented at 37°C for 48 hours. After fermentation, the mixture was sterilized at high temperature. *Lactobacillus plantarum* YYS-EN1 ferments the traditional Chinese medicine, decomposing cellulose and pectin, reducing tannins and phytic acid, thus reducing bitterness and enhancing flavor.

[0174] The traditional Chinese medicine mentioned is not limited to the specific traditional Chinese medicines described in the examples, but includes those listed in the catalog of food and medicine homology.

[0175] The probiotic combination includes, but is not limited to, Lactobacillus plantarum YYS-EN1 powder and Lactobacillus plantarum BXM2 powder. It can also be used alone or in combination with other probiotic powders.

[0176] Among them, Lactobacillus plantarum ( Lactobacillus plantarum BMX2 was deposited on September 6, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.16436.

[0177] Based on the results of the above embodiments, the *Lactobacillus plantarum* YYS-EN1 provided by the present invention has the following properties and effects:

[0178] 1. It has the ability to produce cellulase, with a cellulase activity of 55.21 ± 0.06 U·mL. -1 It can decompose plant cellulose and can be widely used in the development of silage products.

[0179] 2. It possesses pectinase activity, with a pectinase activity of 45.28 ± 0.07 U·mL. -1 It can break down pectin in fruits and vegetables, and can be widely used in the development of juices, vegetables, wines, enzymes and other products to improve product clarity and yield.

[0180] 3. It possesses tanninase activity, with a tanninase activity of 27.55 ± 0.03 U·mL. -1 It can reduce the tannin content in wine and can be widely used in the development of wine and enzyme products to improve the taste of the products.

[0181] 4. It possesses phytase activity, with a phytase activity of 19.79 ± 0.04 U·mL. -1 It can effectively decompose phytic acid and can be widely used in flour and soybean food processing to improve their nutritional and commercial value.

[0182] 5. It can be used in the production of corn nutrient powder to degrade cellulose and phytic acid in corn husks and corn germ, thereby improving the nutritional value of corn nutrient powder and enhancing its taste.

[0183] 6. The strain is derived from rice wine and is a natural probiotic that can be used as a fermentation agent in the preparation of wine, enzymes, feed, etc.

[0184] In summary, compared with the prior art, the *Lactobacillus plantarum* YYS-EN1 provided by this invention has the following beneficial effects:

[0185] The *Lactobacillus plantarum* YYS-EN1 provided by this invention can be used in probiotic products and functional foods. It colonizes the human gut, improving gut microbiota structure and promoting digestion and absorption. It produces cellulase, pectinase, tanninase, and phytase, and can be used in probiotic fermented products (including but not limited to beverages, foods, and feeds). Examples of fermented products include, but are not limited to, the preparation of fermented plant-derived nutrient powders, fermented plant-derived polypeptide powders, fermented plant-derived protein powders, enzymes, and silage.

[0186] Among them, the *Lactobacillus plantarum* YYS-EN1 provided by this invention produces cellulase and pectinase, which can degrade plant cellulose and pectin, and can be applied to the development of fermented plant-derived nutrient powder, fruit juice, vegetable feed and other products; at the same time, this strain has a high capacity for tannin and phytase, and can be widely used in the production of fermented products, such as wine, enzymes and other products, and can significantly reduce the tannin content in the products.

[0187] In summary, it can be specifically applied to functional products that include at least one of the following functions:

[0188] (1) It has the ability to degrade cellulose;

[0189] (2) It has the ability to degrade pectin;

[0190] (3) It has the ability to degrade tannins;

[0191] (4) It has the ability to degrade phytic acid;

[0192] Among them, products with the above (1)-(4) functions include, but are not limited to, decomposing plant fibers and pectin, improving product clarity, etc., and can also be products with other obvious effects based on the degradation and decomposition of plant fibers, pectin, tannins or phytic acid.

[0193] Based on the above characteristics, the *Lactobacillus plantarum* YYS-EN1 provided by this invention has the following beneficial effects:

[0194] The *Lactobacillus plantarum* YYS-EN1 provided by this invention has the ability to produce cellulase, pectinase, tanninase and phytase, and can degrade plant cellulose and pectin.

[0195] It has the ability to produce cellulase, which can degrade cellulose and can be applied to the development of fermented plant-derived nutrient powders, silage products, etc.

[0196] It has the ability to degrade pectinase and can be widely used in the production of products such as fruit juice, vegetables, wine, and enzymes. It can significantly reduce the pectin content in raw materials and improve the clarity and yield of products.

[0197] It has the ability to produce tannins and can be widely used in the production of wine, enzymes and other products. It can reduce the tannin content in wine and improve the taste of the product.

[0198] It has the ability to produce phytase, which can be widely used in flour and soybean food processing to improve the degradation rate of phytic acid and enhance its nutritional and commercial value.

[0199] The *Lactobacillus plantarum* YYS-EN1 provided by this invention produces cellulase, pectinase, tanninase, and phytase, and can be applied to various probiotic fermentation products. These fermentation products include, but are not limited to, fermented plant-derived nutrient powder, fermented plant-derived polypeptide powder, fermented plant-derived protein powder, enzymes, silage, etc., laying the foundation for providing green, healthy, high-nutritional-value, and highly-utilized plant-derived agricultural products.

[0200] It should be noted that:

[0201] (1) Definition:

[0202] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. This application can be used to prepare solid dosage forms such as powders, tablets, and gels, and also to disperse in liquids to prepare liquid dosage forms, including but not limited to the embodiments described herein.

[0203] (2) The relevant prior art means or prior art terms involved in this application:

[0204] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through an analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. x "OD" is the optical density value measured when the wavelength is set to X nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate the density of bacterial cells. The method for measuring the "OD" value is existing technology, and its principle and method will not be described here.

[0205] This article uses the Oxford Cup method as an existing technique, and its process will not be described in detail.

[0206] (3) Application of strains:

[0207] The examples illustrate that *Lactobacillus plantarum* YYS-EN1 can be applied to corn nutrient powder products. According to the above design concept, this strain can be applied to various products containing cellulose and pectin, and can be used in the preparation of fermented products, including fermented plant-derived nutrient powder, fermented plant-derived polypeptide powder, fermented plant-derived protein powder, enzymes, silage, etc., including but not limited to the corn nutrient powder in the examples.

[0208] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum YYS-EN1, characterized in that, The accession number is CGMCC No. 28532.

2. The *Lactobacillus plantarum* YYS-EN1 according to claim 1, characterized in that: It has the ability to produce cellulase, pectinase, tanninase and phytase.

3. The *Lactobacillus plantarum* YYS-EN1 according to claim 1, characterized in that: It has the ability to degrade cellulose, pectin, tannins and phytic acid.

4. A method for producing cellulase, pectinase, tanninase, and phytase, characterized in that: Lactobacillus plantarum YYS-EN1 was inoculated into an enzyme-producing liquid culture medium and cultured at 30-40°C for 48-72 hours. The enzyme-producing liquid culture medium after fermentation was separated into solid and liquid components to obtain a supernatant, which was an enzyme solution containing cellulase, pectinase, tanninase and phytase. Wherein, the plant lactobacillus YYS-EN1 is the plant lactobacillus YYS-EN1 as described in any one of claims 1-3.

5. The application of *Lactobacillus plantarum* YYS-EN1 in the preparation of fermented products, characterized by: The plant lactobacillus YYS-EN1 is the plant lactobacillus YYS-EN1 as described in any one of claims 1-3; the fermentation product includes fermented nutrient powder or silage. The fermented nutrient powder includes fermented plant-derived nutrient powder.

6. A freeze-dried powder, characterized in that: Its components include *Lactobacillus plantarum* YYS-EN1 as described in any one of claims 1-3.

7. A microbial agent, characterized in that: The microbial agent comprises the freeze-dried powder as described in claim 6, or Lactobacillus plantarum YYS-EN1 as described in any one of claims 1-3.

8. The application of *Lactobacillus plantarum* YYS-EN1 in the preparation of probiotic products, characterized by: The plant lactobacillus YYS-EN1 is the plant lactobacillus YYS-EN1 as described in any one of claims 1-3.

9. The application of *Lactobacillus plantarum* YYS-EN1 in the preparation of functional products, characterized by: The functional product includes at least one of the following functions: (1) It has the ability to degrade cellulose; (2) It has the ability to degrade pectin; (3) It has the ability to degrade tannins; (4) It has the ability to degrade phytic acid; Wherein, the plant lactobacillus YYS-EN1 is the plant lactobacillus YYS-EN1 as described in any one of claims 1-3.

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